5 replies to this topic

[shojjahd]

Hi All,

Please can someone help  

I want to express a protein in HEK293 cells via transfection in order to do functional studies (not for purification etc) so I dont think I want it tagged etc etc. But do you use the full length cDNA or the ORF clone? Both are available. I have no clue - please help!!

I am just interested in maximal expression of the active protein and will assay the cells directly for function.

Thanks in advance!

[WHR]

We used to express the ORF only.  Including a Kozak sequence in front of the start codon may helps.

[scolix]

As suggested, use ORF.

Remember transient transfection will give you something like 1000x overexpression of the protein.

[shasa753]

Hi,
sorry to bump in with not-exactly-related to the original question.  But i have a question about transfection of retroviral vector using fugene6.  found an exact post with the exact prob but the link was broken so i couldn't follow on that.  Here's the situation:

Need to transfect an LZRS-ires-egfp (~13kb) plasmid into 293GP cell;
Did:

1. optimizing transfection using (ugDNA:ulFugene) 2:9, 3:9, 4:9, 5:9
2. optimize again using (ugDNA:ulFugene) 2:3, 2:6, 2:9, 2:12

all of these in 6-well plate with 40-50% cell confluency.  The transfection results was very2 low as seen by microscopy while FACS showed it was ~20-30%.  A control transfectin using pEGFPc1 (1ug:9ul) have better results (~80% by microscopy, though funny that FACS says it was just ~40%).

prep of dna:fugene mix and transfection were as follows:

- optimem + fugene (to final vol. 100ul) - incubate RT 5 min,
- optimem/fugene mix + DNA - incubate RT 1 hr
- remove overnight medium from plate and dropwise mix onto cell
- incubate at RT 1 hr with tilting of plate every 15 minutes
- top wells with 2ml complete DMEM medium (10% fcs + antibiotic) and incubate 48hrs before microscopy or facs-ing

Have any idea how else should i troubleshoot the transfection?  Really appreciate for any feedbacks.

Thanks in advance.

[NemomeN]

shasa753, on Mar 1 2009, 07:51 PM, said:

Hi,
sorry to bump in with not-exactly-related to the original question.  But i have a question about transfection of retroviral vector using fugene6.  found an exact post with the exact prob but the link was broken so i couldn't follow on that.  Here's the situation:

Need to transfect an LZRS-ires-egfp (~13kb) plasmid into 293GP cell;
Did:

1. optimizing transfection using (ugDNA:ulFugene) 2:9, 3:9, 4:9, 5:9
2. optimize again using (ugDNA:ulFugene) 2:3, 2:6, 2:9, 2:12

all of these in 6-well plate with 40-50% cell confluency.  The transfection results was very2 low as seen by microscopy while FACS showed it was ~20-30%.  A control transfectin using pEGFPc1 (1ug:9ul) have better results (~80% by microscopy, though funny that FACS says it was just ~40%).

prep of dna:fugene mix and transfection were as follows:

- optimem + fugene (to final vol. 100ul) - incubate RT 5 min,
- optimem/fugene mix + DNA - incubate RT 1 hr
- remove overnight medium from plate and dropwise mix onto cell
- incubate at RT 1 hr with tilting of plate every 15 minutes
- top wells with 2ml complete DMEM medium (10% fcs + antibiotic) and incubate 48hrs before microscopy or facs-ing

Have any idea how else should i troubleshoot the transfection?  Really appreciate for any feedbacks.

Thanks in advance.

Sounds like your DNA is not very clean.  Fugene and 293 cells should be quite easy...normally, I use 2ul of fugene per microgram of DNA...and I use PBS.  I try to stay away from optimem as it does contain some protein (~15ug/ml).  You should see a fine hazy consistency after you add the fugene to the DNA (appears in a couple minutes or so, especially if you are transfecting ug of DNA) ...and do not remove the media before you add the fugene.  Just add it to the media, swirl a few times and that's it.  You actually need the media to disperse the DNA around or you will not get an even transfection.

good luck

[shasa753]

NemomeN, on Mar 2 2009, 04:17 PM, said:

shasa753, on Mar 1 2009, 07:51 PM, said:

Hi,
sorry to bump in with not-exactly-related to the original question.  But i have a question about transfection of retroviral vector using fugene6.  found an exact post with the exact prob but the link was broken so i couldn't follow on that.  Here's the situation:

Need to transfect an LZRS-ires-egfp (~13kb) plasmid into 293GP cell;
Did:

1. optimizing transfection using (ugDNA:ulFugene) 2:9, 3:9, 4:9, 5:9
2. optimize again using (ugDNA:ulFugene) 2:3, 2:6, 2:9, 2:12

all of these in 6-well plate with 40-50% cell confluency.  The transfection results was very2 low as seen by microscopy while FACS showed it was ~20-30%.  A control transfectin using pEGFPc1 (1ug:9ul) have better results (~80% by microscopy, though funny that FACS says it was just ~40%).

prep of dna:fugene mix and transfection were as follows:

- optimem + fugene (to final vol. 100ul) - incubate RT 5 min,
- optimem/fugene mix + DNA - incubate RT 1 hr
- remove overnight medium from plate and dropwise mix onto cell
- incubate at RT 1 hr with tilting of plate every 15 minutes
- top wells with 2ml complete DMEM medium (10% fcs + antibiotic) and incubate 48hrs before microscopy or facs-ing

Have any idea how else should i troubleshoot the transfection?  Really appreciate for any feedbacks.

Thanks in advance.

Sounds like your DNA is not very clean.  Fugene and 293 cells should be quite easy...normally, I use 2ul of fugene per microgram of DNA...and I use PBS.  I try to stay away from optimem as it does contain some protein (~15ug/ml).  You should see a fine hazy consistency after you add the fugene to the DNA (appears in a couple minutes or so, especially if you are transfecting ug of DNA) ...and do not remove the media before you add the fugene.  Just add it to the media, swirl a few times and that's it.  You actually need the media to disperse the DNA around or you will not get an even transfection.

good luck

i doubt it's the DNA.  prepared it used qiagen midiprep and nanodrop 280/260 reading is 1.87.  but using PBS in place of optimem is new.  I'll try this one today hopefully this'll work.

thank you